CHARACTERIZING NEURAL RETICULON 1 AND DETERMINING THE MECHANISM OF MAINTAINING A DIFFUSION BARRIER IN THE AXON INITIAL SEGMENT IN DROSOPHILA MELANOGASTER NEURONS

Open Access
Author:
Luna, Esteban
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Melissa Rolls, Thesis Supervisor
  • Craig Eugene Cameron, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • Drosophila melanogaster
  • neurons
  • diffusion barrier
  • reticulon
  • ankyrin 2
  • FRAP
Abstract:
Drosophila melanogaster neurons provide an excellent model to study proteins associated with neurodegeneration and regeneration. Among the families of proteins associated with axon regeneration is the general endoplasmic reticulum (ER) protein family, reticulon (rtnl). This family is conserved from Drosophila and humans. It has been implicated in limiting the regrowth of axons in the CNS after injury, but their true function, as of yet, has been undiscovered. The mammalian genome contains 4 rtnl genes, while the Drosophila genome only contains 2. Because there appears to be less redundancy in the Drosophila genome, it has been suggested that it may be easier to study rtnl proteins in Drosophila (Wolfe, 2003). In the first part of this study, I hypothesized that rtnl 1 is an ER structural protein. This is because the decrease of NogoA, an isoform of rtnl 4 in mammals, appears to allow axons to regrow past the site of injury while maintaining its shape, which is dependent upon the cytoskeleton (Schwab, 2004). I found that rtnl 1-GFP colocalizes with microtubules, and is less mobile compared to another ER protein, KDEL-GFP, and a general membrane protein, mcd8-GFP in the cell body by utilizing fluorescence recovery after photobleaching (FRAP). I also found that rtnl 1-GFP had approximately the same mobility in the axon initial segment (AIS) as it did in the cell body. Surprisingly, mcd8-GFP exhibited less mobility in the plasma membrane of the AIS compared to itself in the cell body. The second part of the study is dedicated to elucidating the existence of a barrier to diffusion in the plasma membrane of the AIS and its mechanism. A diffusion barrier has been found in the AIS of mammalian neurons, and it has also been found that it is dependent upon the structural protein ankyrin G (ankG) (Nakada et al, 2003). A diffusion barrier has been found in the distal portion of the axon in the plasma membrane in immature Drosophila neurons, but no barrier has been characterized in the AIS similar to that found in mammals (Katsuki et al., 2009). In this study, I found that there is a diffusion barrier in the AIS that does not appear to exist in other parts of the neuron and that the diffusion barrier is dependent on ank2, a structural protein similar to ankG. These studies help characterize rtnl 1 in the Drosophila neural system via confocal microscopy. Moreover, a diffusion barrier is identified in the AIS and its mechanism is partially elucidated, revealing its similarity to the barrier in mammals. This shows that Drosophila neurons are more similar to mammalian neurons than previously believed, and Drosophila can be used as a better model for neural studies.